Estrogen receptors (ERs) mediate the diverse effects of estrogen on the cardiovascular system. The two known ERs, ER1 and ER2, are ligand-activated transcription factors. The transcriptional effects of ERs depend on receptor interacting proteins, including SRC3, a member of the family of co-activator proteins. Commonly, the genomic effects just described are differentiated from more rapid effects that do not require alterations in gene expression and thus are called `non-genomic' effects. The best-studied non-genomic ER pathway in vascular endothelial cells (EC) involves rapid activation of the kinase Akt, and subsequent activation of endothelial nitric oxide synthase (eNOS). eNOS protein abundance is also transcriptionally regulated by ERs. These two signaling pathways, genomic and non-genomic, have largely been considered independent, and the physiologic relevance of the non-genomic pathway has been questioned. We recently identified the protein striatin as an ER1 interacting protein necessary for assembly of cell membrane-associated signaling complexes that mediate non-genomic estrogen-induced eNOS phosphorylation. Initially, we demonstrated that inhibition of ER1-striatin binding blocked rapid activation or Akt and eNOS, but did not inhibit transcriptional activation of an estrogen-response-element driven reporter, supporting that the striatin-ER1 interaction is not required for transcriptional activation by ER1. However, we now report that inhibition of ER1-striatin binding prevents ER1-mediated transcriptional regulation of the eNOS gene, and that the co-activator protein SRC3 in necessary for both non-genomic and genomic regulation of eNOS by ER1. Based on these findings, we propose the central hypothesis of this application, that a multi-protein complex that includes ER1, the scaffolding protein striatin, and the coactivator SRC3 regulates both non-genomic and genomic effects of estrogen in EC and vascular physiology. We propose to test this hypothesis with the following specific aims: Specific Aim 1: To investigate the mechanisms by which striatin and SRC3 regulate rapid (non-genomic) ER-dependent activation of eNOS in ECs, Specific Aim 2: To investigate the mechanisms by which striatin and SRC3 regulate ER-dependent transcriptional (genomic) activation of eNOS in ECs, and Specific Aim 3: To determine the importance of ER-striatin binding in vivo by studying a transgenic mouse that expresses a peptide that disrupts ER1-striatin binding. The results of these studies will provide insight into the importance of non-genomic ER1-mediated signaling in vascular cells and its role in regulating longer-term genomic effects of the receptor. These studies have important implications for furthering our understanding of the effects of estrogen on the vascular system and for developing novel therapeutic agents that act via these signaling pathways.